Mitotic kinesin inhibitors

ABSTRACT

The present invention relates to dihydroisoxazoles that are useful for treating cellular proliferative diseases, for treating disorders associated with KSP kinesin activity, and for inhibiting KSP kinesin. The invention also relates to compositions which comprise these compounds, and methods of using them to treat cancer in mammals.

PRIORITY CLAIM

This application is a §371 application of PCT/US2005/028968 that wasfiled on Aug. 15, 2005, which claims priority from the U.S. ProvisionalApplication No. 60/602,479, filed on Aug. 18, 2004, now expired.

BACKGROUND OF THE INVENTION

This invention relates to dihydroisoxazoles that are inhibitors ofmitotic kinesins (in particular the mitotic kinesin KSP) and are usefulin the treatment of cellular proliferative diseases, for example cancer,hyperplasias, restenosis, cardiac hypertrophy, immune disorders andinflammation.

Among the therapeutic agents used to treat cancer are the taxanes andvinca alkaloids. Taxanes and vinca alkaloids act on microtubules, whichare present in a variety of cellular structures. Microtubules are theprimary structural element of the mitotic spindle. The mitotic spindleis responsible for distribution of replicate copies of the genome toeach of the two daughter cells that result from cell division. It ispresumed that disruption of the mitotic spindle by these drugs resultsin inhibition of cancer cell division, and induction of cancer celldeath. However, microtubules form other types of cellular structures,including tracks for intracellular transport in nerve processes. Becausethese agents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce that drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins that have been identified is KSP. KSP belongsto an evolutionarily conserved kinesin subfamily of plus end directedmicrotubule motors that assemble into bipolar homotetramers consistingof antiparallel homodimers. During mitosis KSP associates withmicrotubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules it the spindle pole.

Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-141 (1996); Blangy, etal., J. Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426], and a fragment of theKSP gene (TRIP5) has been described [Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number LA0372]. Xenopus KSP homologs(Eg5), as well as Drosophila K-LP61 F/KRP 130 have been reported.

Mitotic kinesins are attractive targets for the discovery anddevelopment of novel mitotic chemotherapeutics. Accordingly, it is anobject of the present invention to provide compounds, methods andcompositions useful in the inhibition of KSP, a mitotic kinesin.

SUMMARY OF THE INVENTION

The present invention relates to dihydroisoxazoles that are useful fortreating cellular proliferative diseases, for treating disordersassociated with KSP kinesin activity, and for inhibiting KSP kinesin.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful in the inhibition of mitotickinesins. A first embodiment of the instant invention is a compound asillustrated by Formula I:

wherein:

the dashed line represents an optional double bond;

a is 0 or 1; b is 0 or 1; m is 0, 1 or 2;

R¹, R², R³ and R⁴ are independently selected from: H, CF₃, NO₂,(C═O)_(a)O_(b)(C₁-C₁₀)alkyl, (C═O)_(a)O_(b)(C₂-C₁₀)alkenyl,(C═O)_(a)O_(b)(C₂-C₁₀)alkynyl, (C═O)_(a)O_(b)(C₃-C₈)cycloalkyl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-aryl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-heterocyclyl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-N(R^(d))₂,N(C═O)O_(b)(C₁-C₃)perfluoroalkyl, (C₀-C₆)alkylene-S(O)_(m)R^(a),(C₀-C₆)alkylene-CO₂R^(a), C(O)H, (C₀-C₆)alkylene-CO₂H, andS(O)_(m)N(R^(b))₂; said alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylene and heterocyclyl are optionally substituted with up to threesubstituents selected from R⁷;

R⁷ is independently selected from: H, halogen, CF₃, (C₁-C₆)alkyl,(C═O)_(a)O_(b)(C₁-C₆)alkyl, (C═O)cycloalkyl, (C═O)aryl,(C═O)heterocyclyl, (C₃-C₈)cycloalkyl, aryl, heterocyclyl, OH, oxo, CN,(C₁-C₆)alkyl-OH, (C₁-C₆)alkyl-N(R^(c))₂, N(R^(c))₂, —OP(O)OH₂ and—O(C═O)(C₁-C₆)alkyl;

R^(a) is selected from: (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, aryl andheterocyclyl; said alkyl, cycloalkyl, aryl and heterocyclyl isoptionally substituted with one or more substituents selected from R⁷;

R^(b) is independently selected from: H, (C₁-C₆)alkyl and heterocyclyl;

R^(c) is independently selected from: H and (C₁-C₆)alkyl;

R^(d) is independently selected from: H, O_(a)(C₁-C₆)alkyl,(C₁-C₆)alkyl-heterocyclyl, (C₃-C₈)cycloalkyl, heterocyclyl, said alkyl,cycloalkyl and heterocyclyl optionally substituted with up to threesubstituents selected from R⁷;

R^(z) is selected from H, (C₁-C₆)alkyl, —O(C₁-C₆)alkyl, (C₁-C₆)alkyl-OH,(C₁-C₆)alkyl-N(R^(c))₂ and OH, or R^(z) is absent if the dashed linerepresents a double bond;

or a pharmaceutically acceptable salt or stereoisomer thereof.

A second embodiment of the instant invention is a compound asillustrated by Formula I:

wherein:

-   -   R¹ is selected from: H, CF₃, NO₂, (C═O)_(a)O_(b)(C₁-C₁₀)alkyl,        (C═O)_(a)O_(b)(C₂-C₁₀)alkenyl, (C═O)_(a)O_(b)(C₂-C₁₀)alkynyl,        (C═O)_(a)O_(b)(C₃-C₈)cycloalkyl,        (C═O)_(a)O_(b)(C₀-C₆)alkylene-aryl,        (C═O)_(a)O_(b)(C₀-C₆)alkylene-heterocyclyl,        (C═O)_(a)O_(b)(C₀-C₆)alkylene-N(R^(d))₂,        O_(b)(C₁-C₃)perfluoroalkyl, (C₀-C₆)alkylene-S(O)_(m)R^(a),        (C₀-C₆)alkylene-CO₂R^(a), C(O)H, (C₀-C₆)alkylene-CO₂H, and        S(O)_(m)N(R^(b))₂, said alkyl, alkenyl, alkynyl, cycloalkyl,        aryl, alkylene and heterocyclyl is optionally substituted with        up to three substituents selected from R⁷;

R² is selected from: (C₃-C₈)cycloalkyl, aryl and heterocyclyl; saidcycloalkyl, aryl and heterocyclyl optionally substituted with up tothree substituents selected from R⁷;

R³ is selected from: H, (C₁-C₆)alkyl-N(R^(d))₂,(C₁-C₆)alkyl-heterocyclyl, (C₁-C₆)alkyl-OH, O_(b)(C₁-C₁₀)alkyl,(C₃-C₈)cycloalkyl, heterocyclyl, N(R^(d))₂, (C₁-C₆)alkyl(O)(C₁-C₆)alkyl,said heterocyclyl optionally substituted with up to three substituentsselected from R⁷;

R⁴ is selected from: (C₃-C₈)cycloalkyl, aryl and heterocyclyl; saidcycloalkyl, aryl and heterocyclyl optionally substituted with up tothree substituents selected from R⁷;

R^(z) is selected from: H, (C₁-C₆)alkyl and OH, or R^(z) is absent ifthe dashed line represents a double bond;

and all other substituents and variables are as defined in the firstembodiment;

or a pharmaceutically acceptable salt or stereoisomer thereof

A third embodiment of the instant invention is a compound as illustratedby Formula II:

wherein:

n and p are independently 0, 1, 2, 3, 4 or 5;

A and B are independently selected from: (C₃-C₈)cycloalkyl, aryl andheterocyclyl;

R³ is selected from: H(C₁-C₆)alkyl-N(R^(d))₂, (C₁-C₆)alkyl-heterocyclyl,(C₁-C₆)alkyl-OH, O_(b)(C₁-C₁₀)alkyl, (C₃-C₈)cycloalkyl, heterocyclyl,N(R^(d))₂, (C₁-C₆)alkyl(O)(C₁-C₆)alkyl, said heterocyclyl optionallysubstituted with up to three substituents selected from R⁸;

R⁵ is independently selected from H, CF₃, (C₁-C₆)alkyl-N(R^(c))₂,N(R^(c))₂, (C₁-C₆)alkyl, halogen and OH;

R⁶ is independently selected from: H, (C₁-C₆)alkyl, halogen and OH;

R⁸ is independently selected from H, halogen, (C₁-C₆)alkyl,(C═O)_(a)O_(b)(C₁-C₆)alkyl, (C═O)cycloalkyl, (C═O)aryl,(C═O)heterocyclyl, (C₃-C₈)cycloalkyl, aryl, heterocyclyl, OH, oxo, CN,(C₁-C₆)alkyl-OH, N(R^(c))₂, —OP(O)OH₂ and —O(C═O)(C₁-C₆)alkyl;

and all other substituents and variables are as defined in the secondembodiment;

or a pharmaceutically acceptable salt or stereoisomer thereof.

A fourth embodiment of the instant invention is a compound asillustrated by Formula III:

wherein:

and all other substituents and variables are as defined in the thirdembodiment;

or a pharmaceutically acceptable salt or stereoisomer thereof.

Specific examples of the compounds of the instant invention include:

-   2-benzyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole (1-3);-   2-cyclohexyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole    (1-4);-   5-(2,5-difluorophenyl)-2-isopropyl-3-phenyl-2,3-dihydroisoxazole    (1-5);-   5-(2,5-difluorophenyl)-2-methyl-3-phenyl-2,3-dihydroisoxazole (1-6);-   5-(5-chloro-2-fluorophenyl)-N,3-dimethyl-N-(1-methylpiperidin-4-yl)-3-phenylisoxazole-2(3H)-carboxamide    (2-6);-   2-acetyl-5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole    (2-7);-   5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole    (2-4);-   5-(5-chloro-2-fluorophenyl)-2-(2,2-dimethylpropanoyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole    (2-9);-   5-(5-chloro-2-fluorophenyl)-N-[3-(dimethylamino)propyl]-N,3-diethyl-3-phenylisoxazole-2(3H)-carboxamide    (2-10); and-   2-acetyl-5-(2,5-difluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole    (2-11);    or a pharmaceutically acceptable salt or stereoisomer thereof.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, all such stereoisomers beingincluded in the present invention. In addition, the compounds disclosedherein may exist as tautomers and both tautomeric forms are intended tobe encompassed by the scope of the invention, even though only onetautomeric structure is depicted.

When any variable (e.g. R⁵ and R⁶, etc.) occurs more than one time inany constituent, its definition on each occurrence is independent atevery other occurrence. Also, combinations of substituents and variablesare permissible only if such combinations result in stable compounds.Lines drawn into the ring systems from substituents represent that theindicated bond may be attached to any of the substitutable ring atoms.If the ring system is polycyclic, it is intended that the bond beattached to any of the suitable carbon atoms on the proximal ring only.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrase “optionally substituted with one or more substituents” shouldbe taken to be equivalent to the phrase “optionally substituted with atleast one substituent” and in such cases the preferred embodiment willhave from zero to three substituents.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 carbons in a linear or branched arrangement. For example, “C₁-C₁₀alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. For example,“cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. Inan embodiment of the invention the term “cycloalkyl” includes the groupsdescribed immediately above and further includes monocyclic unsaturatedaliphatic hydrocarbon groups. For example, “cycloalkyl” as defined inthis embodiment includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl,cyclobutenyl and so on.

The term “alkylene” means a hydrocarbon diradical group having thespecified number of carbon atoms. For example, “alkylene” includes—CH₂—, —CH₂CH₂— and the like.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, straight, branched or cyclic,containing from 2 to 10 carbon atoms and at least one carbon to carbondouble bond. Preferably one carbon to carbon double bond is present, andup to four non-aromatic carbon-carbon double bonds may be present. Thus,“C₂-C₆ alkenyl” means an alkenyl radical having from 2 to 6 carbonatoms. Alkenyl groups include ethenyl, propenyl, butenyl,2-methylbutenyl and cyclohexenyl. The straight, branched or cyclicportion of the alkenyl group may contain double bonds and may besubstituted if a substituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon radical straight, branched orcyclic, containing from 2 to 10 carbon atoms and at least one carbon tocarbon triple bond. Up to three carbon-carbon triple bonds may bepresent. Thus, “C₂-C₆ alkynyl” means an alkynyl radical having from 2 to6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl,3-methylbutynyl and so on. The straight, branched or cyclic portion ofthe alkynyl group may contain triple bonds and may be substituted if asubstituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkylene-aryl. If aryl istaken to be phenyl, this definition would include phenyl itself as wellas —CH₂Ph, —CH₂CH₂Ph, —CH(CH₃)CH₂CH(CH₃)Ph, and so on.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where thearyl substituent is bicyclic and, one ring is non-aromatic, it isunderstood that attachment is via the aromatic ring.

The term “heterocycle”¹ or “heterocyclyl” as used herein is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. “Heterocyclyl” therefore includes theabove mentioned heteroaryls, as well as dihydro and tetrahydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, diazepanyl, diazepanonyl, furanyl,imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl,isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl,oxadiazolyl, oxazolyl, oxazolinyl, isoxazolinyl, oxetanyl, pyranyl,pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl,pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazyl,azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro (Cl), fluoro (F), bromo (Br) andiodo (I).

In an embodiment, A is (C₃-C₈)cycloalkyl, aryl or heterocyclyl.

In another embodiment A is phenyl.

In an embodiment, B is (C₃-C₈)cycloalkyl, aryl or heterocyclyl.

In another embodiment B is phenyl.

In an embodiment, the dashed line represents a double bond.

In an embodiment, n is 1, 2 or 3.

In another embodiment, n is 1 or 2.

In another embodiment, n is 2.

In an embodiment, p is 0, 1, 2 or 3.

In another embodiment, p is 0, 1 or 2.

In another embodiment, p is 0.

In an embodiment, R¹ is selected from: H, CF₃, NO₂,(C═O)_(a)O_(b)(C₁-C₁₀)alkyl, (C═O)_(a)O_(b)(C₂-C₁₀)alkenyl,(C═O)_(a)O_(b)(C₂-C₁₀)alkynyl, (C═O)_(a)O_(b)(C₃-C₈)cycloalkyl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-aryl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-heterocyclyl,(C═O)_(a)O_(b)(C₀-C₆)alkylene-N(R^(d))₂, O_(b)(C₁-C₃)perfluoroalkyl,(C₀-C₆)alkylene-S(O)_(m)R^(a), (C₀-C₆)alkylene-CO₂R^(a), C(O)H,(C₀-C₆)alkylene-CO₂H, and S(O)_(m)N(R^(b))₂; said alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkylene and heterocyclyl are optionallysubstituted with up to three substituents selected from R⁷.

In another embodiment, R¹ is selected from: H, (C═O)_(a)(C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C═O)N(R^(d))₂, aryl and heterocyclyl; said alkyl,cycloalkyl, aryl and heterocyclyl are optionally substituted with 1-3substituents selected from R⁷.

In another embodiment, R¹ is selected from: H, (C═O)_(a)(C₁-C₆)alkyl,(C₁-C₆)alkyl-phenyl, (C₃-C₈)cycloalkyl and (C═O)N(R^(d))₂.

In an embodiment, R² is selected from: (C₃-C₈)cycloalkyl, aryl andheterocyclyl; said cycloalkyl, aryl and heterocyclyl optionallysubstituted with up to three substituents selected from R⁷.

In another embodiment, R² is phenyl, optionally substituted with 1-3substituents selected from halo.

In another embodiment, R² is:

In an embodiment, R³ is selected from: H, (C₁-C₆)alkyl-N(R^(d))₂,(C₁-C₆)alkyl-heterocyclyl, (C₁-C₆)alkyl-OH, O_(b)(C₁-C₁₀)alkyl,(C₃-C₈)cycloalkyl, heterocyclyl, N(R^(d))₂, (C₁-C₆)alkyl(O)(C₁-C₆)alkyl,said heterocyclyl optionally substituted with up to three substituentsselected from R⁸.

In another embodiment, R³ is selected from: (C₁-C₆)alkyl-N(R^(d))₂,(C₁-C₆)alkyl-heterocyclyl and (C₁-C₆)alkyl-OH, said heterocyclyloptionally substituted with up to three substituents selected from R⁸.

In another embodiment, when R³ is (C₁-C₆)alkyl-heterocyclyl, saidheterocyclyl is selected from:

In another embodiment, R³ is H and (C₁-C₆)alkyl.

In another embodiment, R³ is H.

In an embodiment, R⁴ is selected from: (C₃-C₈)cycloalkyl, aryl andheterocyclyl; said cycloalkyl, aryl and heterocyclyl optionallysubstituted with up to three substituents selected from R⁷.

In another embodiment, R⁴ is phenyl.

In an embodiment, R⁵ is independently selected from: H, CF₃,(C₁-C₆)alkyl-N(R^(c))₂, N(R^(c))², (C₁-C₆)alkyl, halogen and OH.

In another embodiment, R⁵ is halogen.

In another embodiment, R⁵ is F.

In an embodiment, R⁷ is independently selected from: H, halogen, CF₃,(C₁-C₆)alkyl, (C═O)_(a)O_(b)(C₁-C₆)alkyl, (C═O)cycloalkyl, (C═O)aryl,(C═O)heterocyclyl, (C₃-C₈)cycloalkyl, aryl, heterocyclyl, OH, oxo, CN,(C₁-C₆)alkyl-OH, (C₁-C₆)alkyl-N(R^(c))₂, N(R^(c))₂, —OP(O)OH₂ and—O(C═O)(C₁-C₆)alkyl.

In another embodiment, R⁷ is independently selected from: phenyl,halogen, CF₃, (C₁-C₆)alkyl, (C₁-C₆)alkyl-N(R^(c))₂, and N(R^(c))₂.

In an embodiment, R⁸ is independently selected from: H, halogen,(C₁-C₆)alkyl, (C═O)_(a)O_(b)(C₁-C₆)alkyl, (C═O)cycloalkyl, (C═O)aryl,(C═O)heterocyclyl, (C₃-C₈)cycloalkyl, aryl, heterocyclyl, OH, oxo, CN,(C₁-C₆)alkyl-OH, N(R^(c))₂, —OP(O)OH₂ and —O(C═O)(C₁-C₆)alkyl.

In another embodiment, R⁸ is independently selected from: N(R^(c))₂,(C₁-C₆)alkyl, OH, halogen, oxo and (C₁-C₆)alkyl-OH.

In an embodiment, R^(b) is independently selected from: H and(C₁-C₆)alkyl.

In an embodiment, R^(d) is independently selected from: H,O_(a)(C₁-C₆)alkyl, (C₁-C₆)alkyl-heterocyclyl, (C₃-C₈)cycloalkyl,heterocyclyl, said alkyl, cycloalkyl and heterocyclyl optionallysubstituted with up to three substituents selected from R⁷.

In another embodiment, R^(d) is independently selected from: H and(C₁-C₆)alkyl.

Included in the instant invention is the free form of compounds ofFormula I, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the specific compounds exemplified hereinare the protonated salts of amine compounds. The term “free form” refersto the amine compounds in non-salt form. The encompassedpharmaceutically acceptable salts not only include the salts exemplifiedfor the specific compounds described herein, but also all the typicalpharmaceutically acceptable salts of the free form of compounds ofFormula I. The free form of the specific salt compounds described may beisolated using techniques known in the art. For example, the free formmay be regenerated by treating the salt with a suitable dilute aqueousbase solution such as dilute aqueous NaOH, potassium carbonate, ammoniaand sodium bicarbonate. The free forms may differ from their respectivesalt forms somewhat in certain physical properties, such as solubilityin polar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant compounds can besynthesized from the compounds of this invention which contain a basicor acidic moiety by conventional chemical methods. Generally, the saltsof the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the compounds of thisinvention include the conventional non-toxic salts of the compounds ofthis invention as formed by reacting a basic instant compound with aninorganic or organic acid. For example, conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, aswell as salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroaceticand the like.

When the compound of the present invention is acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

The compounds of this invention may be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature or exemplified in theexperimental procedures. The illustrative schemes below, therefore, arenot limited by the compounds listed or by any particular substituentsemployed for illustrative purposes. Substituent numbering as shown inthe schemes does not necessarily correlate to that used in the claimsand often, for clarity, a single substituent is shown attached to thecompound where multiple substituents are allowed under the definitionsof Formula I hereinabove.

Reaction Schemes

In one embodiment (Scheme A), substituted chalcones (A-1) can becombined with alkyl-hydroxylamines (A-2) to form 5-hydroxyisoxazolines(A-3). By the action of MsCl and triethylamine, elimination will occurto give dihydroisoxazoles (A-4). In the case where R¹ is a removablegroup, the unprotected dihydroisoxazole can be produced (A-5). Followingtreatment with phosgene to install a carbonyl, the intermediatecarbamoyl chloride (A-6) can be appended with a variety of amines togive dihydroisoxazole ureas (A-7). Similarly, unprotecteddihydroisoxazole (A-5) can be acylated to produce dihydroisoxazoleamides (A-8).

In another embodiment (Scheme B), substituted chalcones (B-3) can besynthesized wherein OPG is a protected hydroxylalkyl chain from thecorresponding alkyne and aryl Weinreb-amide, followed by conjugateaddition of a second aryl group. The chalcones can be converted toisoxazolines (B-5) by the same manner described in Scheme A. The oxygenprotecting group can be removed to reveal the free alcohol (B-6). Inaddition, this alcohol can be oxidized to aldehyde (B-7) by Dess-Martinperiodinane and then subjected to reductive amination to give amines totype (B-8). The nitrogen group can then be further manipulated by TFAremoval and subsequent acylation to give compounds of type (B-10).

Utility

The compounds of the invention find use in a variety of applications. Aswill be appreciated by those skilled in the art, mitosis may be alteredin a variety of ways; that is, one can affect mitosis either byincreasing or decreasing the activity of a component in the mitoticpathway. Stated differently, mitosis may be affected (e.g., disrupted)by disturbing equilibrium, either by inhibiting or activating certaincomponents. Similar approaches may be used to alter meiosis.

In an embodiment, the compounds of the invention are used to modulatemitotic spindle formation, thus causing prolonged cell cycle arrest inmitosis. By “modulate” herein is meant altering mitotic spindleformation, including increasing and decreasing spindle formation. By“mitotic spindle formation” herein is meant organization of microtubulesinto bipolar structures by mitotic kinesins. By “mitotic spindledysfunction” herein is meant mitotic arrest and monopolar spindleformation.

The compounds of the invention are useful to bind to and/or modulate theactivity of a mitotic kinesin. In an embodiment, the mitotic kinesin isa member of the bimC subfamily of mitotic kinesins (as described in U.S.Pat. No. 6,284,480, column 5). In a further embodiment, the mitotickinesin is human KSP, although the activity of mitotic kinesins fromother organisms may also be modulated by the compounds of the presentinvention. In this context, modulate means either increasing ordecreasing spindle pole separation, causing malformation, i.e.,splaying, of mitotic spindle poles, or otherwise causing morphologicalperturbation of the mitotic spindle. Also included within the definitionof KSP for these purposes are variants and/or fragments of KSP. Inaddition, other mitotic kinesins may be inhibited by the compounds ofthe present invention.

The compounds of the invention are used to treat cellular proliferationdiseases. Disease states which can be treated by the methods andcompositions provided herein include, but are not limited to, cancer(further discussed below), autoimmune disease, arthritis, graftrejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper- or hypoproliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly, as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals which are afflicted or may eventually become afflicted withany one of these disorders or states.

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc. Inparticular, cancers that may be treated by the compounds, compositionsand methods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinomaa), alveolar (bronchiolar) carcinoma,bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma,mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

The compounds of the invention are also useful in preparing a medicamentthat is useful in treating the cellular proliferation diseases above, inparticular cancer.

The compounds of the instant invention may also be useful as antifungalagents, by modulating the activity of the fungal members of the bimCkinesin subgroup, as is described in U.S. Pat. No. 6,284,480.

The compounds of the invention are also useful in preparing a medicamentthat is useful in treating the diseases described above, in particularcancer.

The compounds of this invention may be administered to mammals,preferably humans, either alone or in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintravenous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropyl-methylcellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate butyrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of Formula I may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula I are employed. For purposes ofthis application, topical application shall include mouth washes andgargles.

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Compounds of the presentinvention may also be delivered as a suppository employing bases such ascocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, sex and response of the individual patient, as well as theseverity of the patients symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

The instant compounds are also useful in combination with knowntherapeutic agents and anti-cancer agents. For example, instantcompounds are useful in combination with known anti-cancer agents.Combinations of the presently disclosed compounds with other anti-canceror chemotherapeutic agents are within the scope of the invention.Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6^(th) edition(Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Such anti-cancer agents include, but arenot limited to, the following: estrogen receptor modulators, androgenreceptor modulators, retinoid receptor modulators, cytotoxic/cytostaticagents, antiproliferative agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents and agents that interfere with cell cycle checkpoints.The instant compounds are particularly useful when co-administered withradiation therapy.

In an embodiment, the instant compounds are also useful in combinationwith known anti-cancer agents including the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic agents, antiproliferative agents, prenyl-protein transferaseinhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors,reverse transcriptase inhibitors, and other angiogenesis inhibitors.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and, abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylomithine, ILX23-7553, trans-N-4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell mytosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, inhibitors of histonedeacetylase, inhibitors of kinases involved in mitotic progression,antimetabolites; biological response modifiers; hormonal/anti-hormonaltherapeutic agents, haematopoietic growth factors, monoclonal antibodytargeted therapeutic agents, topoisomerase inhibitors, proteasomeinhibitors and ubiquitin ligase inhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteasome inhibitors include but are not limited tolactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768, WO01/98278, WO 03/050,064, WO 03/050,122, WO 03/049,527, WO 03/049,679, WO03/049,678 and WO 03/39460 and pending PCT Appl. Nos. US03/06403 (filedMar. 4, 2003), US03/15861 (filed May 19, 2003), US03/15810 (filed May19, 2003), US03/18482 (filed Jun. 12, 2003) and US03/18694 (filed Jun.12, 2003). In an embodiment inhibitors of mitotic kinesins include, butare not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors ofCENP-E, inhibitors of MCAK, inhibitors of Kif14, inhibitors of Mphosph1and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are notlimited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Furtherreference to other histone deacetylase inhibitors may be found in thefollowing manuscript; Miller, T. A. et al. J. Med. Chem.46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structuralformulas of these and additional HMG-CoA reductase inhibitors that maybe used in the instant methods are described at page 87 of M. Yalpani,“Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb.1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoAreductase inhibitor as used herein includes all pharmaceuticallyacceptable lactone and open-acid forms (i.e., where the lactone ring isopened to form the free acid) as well as salt and ester forms ofcompounds which have HMG-CoA reductase inhibitory activity, and thereforthe use of such salts, esters, open-acid and lactone forms is includedwithin the scope of this invention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-1), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, P. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in PCT Publication WO03/013,526 and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Inhibitors of cell proliferation and survival signaling pathway” referto pharmaceutical agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of inhibitors of EGFR (for example gefitinib anderlotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of cytokine receptors, inhibitors of MET, inhibitors ofPI3K (for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in (WO 03/086404, WO03/086403, WO 03/086394, WO 03/086279, WO 02/083675, WO 02/083139, WO02/083140 and WO 02/083138), inhibitors of Raf kinase (for exampleBAY-43-9006), inhibitors of MEK (for example CI-1040 and PD-098059) andinhibitors of mTOR (for example Wyeth CCI-779 and Ariad AP23573). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

“Apoptosis inducing agents” include activators of TNF receptor familymembers (including the TRAIL receptors).

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, U.S. Pat. No.5,861,419, U.S. Pat. No. 6,001,843, U.S. Pat. No. 6,020,343, U.S. Pat.No. 5,409,944, U.S. Pat. No. 5,436,265, U.S. Pat. No. 5,536,752, U.S.Pat. No. 5,550,142, U.S. Pat. No. 5,604,260, U.S. Pat. No. 5,698,584,U.S. Pat. No. 5,710,140, WO 94/15932, U.S. Pat. No. 5,344,991, U.S. Pat.No. 5,134,142, U.S. Pat. No. 5,380,738, U.S. Pat. No. 5,393,790, U.S.Pat. No. 5,466,823, U.S. Pat. No. 5,633,272, and U.S. Pat. No.5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are: 3-phenyl-4-(4-methylsulfonyl)phenyl)-2-(5H)-furanone;and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; ora pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the αvβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazo-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chloropheny-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999; 274:9116-9121; Invest. Opthalmol. Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRP552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am J Hum Genet 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), andinterferon gamma (J. Immunol. 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that may result upon administration ofthe instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270; 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93100330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96129326, 96/29328, 96/31214, 96132385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing cancer, including bone cancer, in combination withbisphosphonates (understood to include bisphosphonates, diphosphonates,bisphosphonic acids and diphosphonic acids). Examples of bisphosphonatesinclude but are not limited to: etidronate (Didronel), pamidronate(Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate(Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate,EB-1053, minodronate, neridronate, piridronate and tiludronate includingany and all pharmaceutically acceptable salts, derivatives, hydrates andmixtures thereof.

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: an estrogen receptor modulator, an androgen receptormodulator, retinoid receptor modulator, a cytotoxic/cytostatic agent, anantiproliferative agent, a prenyl-protein transferase inhibitor, anHMG-CoA reductase inhibitor, an HIV protease inhibitor a reversetranscriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, aPPAR-δ agonist, an inhibitor of inherent multidrug resistance, ananti-emetic agent, an agent useful in the treatment of anemia, an agentuseful in the treatment of neutropenia, an immunologic enhancing drug,an inhibitor of cell proliferation and survival signaling, an agent thatinterfers with a cell cycle checkpoint, an apoptosis inducing agent anda bisphosphonate.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

In an embodiment, the angiogenesis inhibitor to be used as the secondcompound is selected from a tyrosine kinase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an MP(matrix metalloprotease inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, or an antibody to VEGF. In an embodiment, the estrogenreceptor modulator is tamoxifen or raloxifene.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of Formula I in combination with radiation therapy and/or incombination with a compound selected from: an estrogen receptormodulator, an androgen receptor modulator, retinoid receptor modulator,a cytotoxic/cytostatic agent, an antiproliferative agent, aprenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, anHIV protease inhibitor, a reverse transcriptase inhibitor, anangiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitorof inherent multidrug resistance, an anti-emetic agent, an agent usefulin the treatment of anemia, an agent useful in the treatment ofneutropenia, an immunologic-enhancing drug, an inhibitor of cellproliferation and survival signaling, an agent that interfers with acell cycle checkpoint, an apoptosis inducing agent and a bisphosphonate.

And yet another embodiment of the invention is a method of treatingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with paclitaxel ortrastuzumab.

The invention further encompasses a method of treating or preventingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with a COX-2 inhibitor.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of Formula I and a compound selectedfrom: an estrogen receptor modulator, an androgen receptor modulator, aretinoid receptor modulator, a cytotoxic/cytostatic agent, anantiproliferative agent, a prenyl-protein transferase inhibitor, anHMG-CoA reductase inhibitor, an HIV protease inhibitor, a reversetranscriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, aPPAR-δ agonist; an inhibitor of cell proliferation and survivalsignaling, an agent that interfers with a cell cycle checkpoint, anapoptosis inducing agent and a bisphosphonate.

These and other aspects of the invention will be apparent from theteachings contained herein.

Abbreviations used in the description of the chemistry and in theExamples that follow are: 9-BBN (9-borabicyclo[3.3.1]nonane); AcOH(acetic acid); DCE (dichloromethane); Dess-Martin Periodinane(1,1,1-tris(aceteloxy)-1,1-benziodoxol-3-(1H)-one); DIBAL-H(diisobutylaluminum hydride); DIEA (diisopropylethylamine); DME(ethylene glycol dimethyl ether); DMF (dimethylformamide); DMSO(dimethyl sulfoxide); DTT (dithiothreitol); EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); EtOAc(ethyl acetate); FACS (fluorescence activated cell sorting); FITC(Fluorescein isothiocyanate); HOBt (1-hydroxybenzotriazole); IPTG(Isopropyl-beta-D-thiogalactopyranoside); LDA (lithiumdiisopropylamide); LHMDS (lithium hexamethyldisilazide); mCPBA(m-chloroperoxybenzoic acid); MS (mass spectrometry); MsCl(methanesulfonyl chloride); NaHMDS (sodium bistrimethylsilylamide); NMR(nuclear magnetic resonance); PMSF (phenylmethylsulphonyl fluoride);PyBop (1H-1,2,3-benzotriazol-1-yloxy)(tripyrrolidin-1-yl)phosphoniumhexafluorophosphate); Rochelle's salt (potassium sodium tartrate); SiO₂(silica gel); TBAI (tetra-n-butylammonium iodide); TEA (triethyl amine);THP (tetrahydrofuran); TFA (trifluoroacteic acid); TMSCN(trimethylsilylcyanide); TsCl (toluenesulfonyl chloride) and Weinrebamide (N-methyl-N-methoxy amide).

EXAMPLES

2-benzyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole (1-3)

A mixture of (2E)-1-(2,5-difluorophenyl)-3-phenylprop-2-en-1-one (1-1)(500 mg, 2.05 mmol, 1 equiv) and N-benzylhydroxylamine hydrochloride(327 mg, 2.05 mmol, 1.00 equiv) was heated at reflux in ethanol (10 mL)for 4 h. Additional N-benzylhydroxylamine hydrochloride (327 mg, 2.05mmol, 1.00 equiv) was added and the mixture was heated at reflux for 16h. The reaction mixture was partitioned between saturated sodiumbicarbonate solution (50 mL) and ethyl acetate (2×50 mL). The combinedorganic layers were dried over sodium sulfate and concentrated. Theresidue was purified by flash column chromatography (silica, 100%hexanes initially, grading to 100% EtOAc) to give2-benzyl-5-(2,5-difluorophenyl)-3-phenylisoxazolidin-5-ol (1-2); LRMSm/z (M+H) 368.3 found, 368.1 required. Methanesulfonyl chloride (0.093uL, 1.20 mmol, 1.11 equiv) was added to a solution of (1-2) (400 mg,1.09 mmol, 1 equiv) and triethylamine (0.455 mL, 3.27 mmol, 3.00 equiv)in dichloromethane (30 mL) at 0° C. and the resultant mixture was warmedto 23° C. and stirred for 30 min. The reaction mixture was partitionedbetween saturated sodium bicarbonate solution (50 mL) anddichloromethane (2×50 mL). The combined organic layers were dried oversodium sulfate and concentrated. The residue was purified by flashcolumn chromatography (silica, 100% hexanes initially, grading to 100%EtOAc) to give2-benzyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole (1-3) as awhite solid. ¹H NMR (300 MHz, CDCl₃) δ 7.46-7.21 (m, 11H), 7.02 (m, 2H),5.76 (t, 1H, J=3.0 Hz), 5.12 (d, 11H, J=2.8 Hz), 4.41 (d, 1H, J=12.8Hz), 4.10 (d, 1H, J=12.8 Hz). LRMS m/z (M+H) 350.3 found, 350.1required.

The following examples were made by simple modifications of the aboveprocedure.

Cmpd Structure Name LRMS m/z (M + H) 1-4

2-cyclohexyl-5-(2,5- difluorophenyl)-3-phenyl-2,3- dihydroisoxazole LRMSm/z (M + H) 342.3 found, 342.2 required. 1-5

5-(2,5-difluorophenyl)-2- isopropyl-3-phenyl-2,3- dihydroisoxazole LRMSm/z (M + H) 302.2 found, 302.1 required 1-6

5-(2,5-difluorophenyl)-2- methyl-3-phenyl-2, 3-dihydroisoxazole LRMS m/z(M + H) 274.2 found, 274.1 required.

5-(5-chloro-2-fluorophenyl)-N,3-dimethyl-N-(1-methylpiperidin-4-yl)-3-phenylisoxazole-2(3H)-carboxamide(2-6)5-(5-chloro-2-fluorophenyl)-2-(2,4-dimethoxybenzyl)-3-methyl-3-phenylisoxazolidin-5-ol(2-2) and5-(5-chloro-2-fluorophenyl)-2-(2,4-dimethoxybenzyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole(2-3)

1-(5-chloro-2-fluorophenyl)-3-phenylbut-2-en-1-one (2-1, mixture of Eand Z isomers, 1.2 g, 4.36 mmol) was dissolved in anhydrous CHCl₃ (50mL) under N₂ and treated with 2,4-dimethoxybenzylhydroxylamine (1.6 g,8.73 mmol) and the reaction stiffed 2 h at 25° C. (2-2). AnhydrousCH₂Cl₂ (150 mL) was added and the reaction was cooled to −10° C.Triethylamine (3.0 mL, 21.8 mmol) was added to the reaction dropwisefollowed by MsCl (1.5 mL, 17.5 mmol) and the reaction was observed tobecome reddish-brown. The reaction was stirred 30 min, and partitionedbetween EtOAc (500 mL) and sat. aq. NaHCO₃ (500 mL). The organic layerwas collected, dried (MgSO₄), filtered and concentrated under reducedpressure. Flash column chromatography (120 g RediSep SiO₂ column, 0-50%EtOAc/hexanes gradient) provided pure5-(5-chloro-2-fluorophenyl)-2-(2,4-dimethoxybenzyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole(2-3). ¹H NMR (300 MHz, CDCl₃) δ 7.61 (m, 2H), 7.44 (dd, J=6.7, 2.4 Hz,1H), 7.40-7.26 (m, 3H), 7.22 (m, 1H), 7.03 (dd, J=10.3, 8.8 Hz, 1H),6.50 (dd, J=8.2, 2.5 Hz, 1H), 6.44 (d, J=2.4 Hz, 1H), 5.90 (d, J=2.4 Hz,1H), 3.81 (s, 3H), 3.78 (m 2H), 3.71 (s, 3H), 1.78 (s, 3H). LRMS m/z(M+H) 440.3 found, 440.9 required.

5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole (2-4)

To a solution of5-(5-chloro-2-fluorophenyl)-2-(2,4-dimethoxybenzyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole(2-3, 0.88 g, 1.99 mmol) in anhydrous CH₂Cl₂ (5 mL) was addedtrifluoroacetic acid (5 mL) and the resulting purple colored reactionsat for 7 h. 2M Na₂CO₃ was added until the color changed from purple topeach, and the reaction was immediately extracted with CH₂Cl₂, driedwith MgSO₄, filtered and concentrated to provide crude5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole(2-4). This crude material was suitable for further transformations. ¹HNMR (300 MHz, CDCl₃) δ 7.56 (m, 3H), 7.36 (m, 3H), 7.25 (m, 1H), 7.05(app t, J=9.6 Hz), 6.20 (br s, 1H), 6.03 (d, J=3.4 Hz, 1H), 1.78 (s,3H). LRMS see table.

5-(5-chloro-2-fluorophenyl)-N,3-dimethyl-N-1-methylpiperidin-4-yl)-3-phenylisoxazole-2(3H)-carboxamide(2-6)

To a solution of crude5-5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole (2-4,0.20 g, 0.69 mmol) and diisopropylethylamine (0.24 mL, 1.4 mmol) inanhydrous THF (3.0 mL) at −5° C. was added phosgene (0.44 mL, 0.83 mmol)and the reaction stirred at 25° C. for 2 h. The reaction was thendiluted with EtOAc (10 mL) and washed with sat. aqueous NH₄Cl (10 mL).The organic layer was dried over MgSO₄, filtered and concentrated toprovide (2-5) which was not characterized. Crude (2-5) was dissolved inanhydrous THF (5 mL) and treated with N-methyl-4-methylaminopiperidine(0.53 g, 4.1 mmol). The reaction was stirred 12 h at 25° C., and thenpartitioned between EtOAc and NH₄Cl. The organic layers were also washedwith NaHCO₃, dried with MgSO₄ and filtered. The organic layer wasconcentrated under reduced pressure, and the crude was purified byreverse phase HPCL (C8 ODS column, 5-95% CH₃CN/H₂O with 0.1% TFAgradient) to yield 5-(5-chloro-2-fluorophenyl)-N,3dimethyl-N-(1-methylpiperidin-4-yl)-3-phenylisoxazole-2(3H)-carboxamide(2-6). ¹H NMR (300 MHz, CDCl₃) δ 7.68 (dd, J=6.2, 2.6 Hz, 11), 7.60 (m,1H), 7.60 (d, J=7.0 Hz, 1H), 7.40-7.30 (m, 2H), 7.34 (d, J=7.1 Hz, 1H),7.25 obscured by CHCl₃ (m, 1H), 7.02 (dd, J=10.3, 8.7 Hz, 1H), 5.65 (d,J=5.6 Hz, 1H), 3.82 (m, 1H), 2.81 (m, 2H), 2.74 (s, 3H), 2.23 (s, 3H),1.93 (s, 3H), 1.82 (m, 1H), 1.64 (m, 4H), 1.24 (m, 1H). LRMS m/z (M+H)444.5 found, 444.9 required.

Cmpd Structure Name LRMS m/z (M + H) 2-7 

2-acetyl-5-(5-chloro-2- fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole LRMS m/z (M + H) 332.2 found, 332.1 required. 2-4 

5-(5-chloro-2-fluorophenyl)-3- methyl-3-phenyl-2,3- dihydroisoxazoleLRMS m/z (M + H) 290.2 found, 290.1 required 2-9 

5-(5-chloro-2-fluorophenyl)-2- (2,2-dimethylpropanoyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole LRMS m/z (M + H) 374.3 found, 347.1required. 2-10

5-(5-chloro-2-fluorophenyl)-N-[3- (dimethylamino)propyl]-N,3-dimethyl-3-phenylisoxazole- 2(3H)-carboxamide LRMS m/z (M + H) 432.5found, 432.2 required. 2-11

2-acetyl-5-(2,5-difluorophenyl)-3- methyl-3-phenyl-2,3- dihydroisoxazoleLRMS m/z (M + H) 316.2 found, 316.1 required.

Assays

The compounds of the instant invention described in the Examples weretested by the assays described below and were found to have kinaseinhibitory activity. Other assays are known in the literature and couldbe readily performed by those of skill in the art (see, for example, PCIPublication WO 01/30768, May 3, 2001, pages 18-22).

I. Kinesin ATPase In Vitro Assay

Cloning and expression of human poly-histidine tagged KSP motor domain(KSP(367H)): Plasmids for the expression of the human KSP motor domainconstruct were cloned by PCR using a pBluescript full length human KSPconstruct (Blangy et al., Cell, vol. 83, pp 1159-1169, 1995) as atemplate. The N-terminal primer5′-GCAACGATTAATATGGCGTCGCAGCCAAATTCGTCTGCGAAG (SEQ.ID.NO.: 1) and theC-terminal primer 5′-GCAACGCTCGAGTCAGTGATGATGGTGGTGATGCGATTCACTTCAGGCTTATTCAATAT (SEQ.ID.NO.: 2) were used toamplify the motor domain and the neck linker region. The PCR productswere digested with AseI and XhoI, ligated into the NdeI/XhoI digestionproduct of pRSETa (Invitrogen) and transformed into E. coli BL21 (DE3).

Cells were grown at 37° C. to an OD₆₀₀ of 0.5. After cooling the cultureto room temperature expression of KSP was induced with 100 μM IPTG andincubation was continued overnight. Cells were pelleted bycentrifugation and washed once with ice-cold PBS. Pellets wereflash-frozen and stored −80° C.

Protein Purification: Cell pellets were thawed on ice and resuspended inlysis buffer (50 mM K-HEPES, pH 8.0, 250 mM KCl, 0.1% Tween, 10 mMimidazole, 0.5 mM Mg-ATP, 1 mM PMSF, 2 mM benzimidine, 1× completeprotease inhibitor cocktail (Roche)). Cell suspensions were incubatedwith 1 mg/ml lysozyme and 5 mM β-mercaptoethanol on ice for 10 minutes,followed by sonication (3×30 sec). All subsequent procedures wereperformed at 4° C. Lysates were centrifuged at 40,000×g for 40 minutes.Supernatants were diluted and loaded onto an SP Sepharose column(Pharmacia, 5 ml cartridge) in buffer A (50 mM K-HEPES, pH 6.8, 1 mMMgCl₂, 1 mM EGTA, 10 μM Mg-ATP, 1 mM DTT) and eluted with a 0 to 750 mMKCl gradient in buffer A. Fractions containing KSP were pooled andincubated with Ni-NTA resin (Qiagen) for one hour. The resin was washedthree times with buffer B (Lysis buffer minus PMSF and proteaseinhibitor cocktail), followed by three 15-minute incubations and washeswith buffer B. Finally, the resin was incubated and washed for 15minutes three times with buffer C (same as buffer B except for pH 6.0)and poured into a column. KSP was eluted with elution buffer (identicalto buffer B except for 150 mM KCl and 250 mM imidazole). KSP-containingfractions were pooled, made 10% in sucrose, and stored at −80° C.

Microtubules are prepared from tubulin isolated from bovine brain.Purified tubulin (>97% MAP-free) at 1 mg/ml is polymerized at 37° C. inthe presence of 10 μM paclitaxel, 1 mM DTT, 1 mM GTP in BRB80 buffer (80mM K-PIPES, 1 mM EGTA, 1 mM MgCl₂ at pH 6.8). The resulting microtubulesare separated from non-polymerized tubulin by ultracentrifugation andremoval of the supernatant. The pellet, containing the microtubules, isgently resuspended in 10 μM paclitaxel, 1 mM DTT, 50 μg/ml ampicillin,and 5 μg/ml chloramphenicol in BRB80.

The kinesin motor domain is incubated with microtubules, 1 mM ATP (1:1MgCl₂: Na-ATP), and compound at 23° C. in buffer containing 80 mMK-HEPES (pH 7.0), 1 mM EGTA, 1 mM DTT, 1 mM MgCl₂, and 50 mM KCl. Thereaction is terminated by a 2-10 fold dilution with a final buffercomposition of 80 mM HEPES and 50 mM EDTA. Free phosphate from the ATPhydrolysis reaction is measured via a quinaldine red/ammonium molybdateassay by adding 150 μl of quench C buffer containing a 2:1 ratio ofquench A:quench B. Quench A contains 0.1 mg/ml quinaldine red and 0.14%polyvinyl alcohol; quench B contains 12.3 mM ammonium molybdatetetrahydrate in 1.15 M sulfuric acid. The reaction is incubated for 10minutes at 23° C., and the absorbance of the phosphomolybdate complex ismeasured at 540 nm.

The compounds shown in the Examples were tested in the above assay andfound to have an IC₅₀≦30 μM.

II. Cell Proliferation Assay

Cells are plated in 96-well tissue culture dishes at densities thatallow for logarithmic growth over the course of 24, 48, and 72 hours andallowed to adhere overnight. The following day, compounds are added in a10-point, one-half log titration to all plates. Each titration series isperformed in triplicate, and a constant DMSO concentration of 0.1% ismaintained throughout the assay. Controls of 0.1% DMSO alone are alsoincluded. Each compound dilution series is made in media without serum.The final concentration of serum in the assay is 5% in a 200 μL volumeof media. Twenty microliters of Alamar blue staining reagent is added toeach sample and control well on the titration plate at 24, 48, or 72hours following the addition of drug and returned to incubation at 37°C. Alamar blue fluorescence is analyzed 6-12 hours later on a CytoFluorII plate reader using 530-560 nanometer wavelength excitation, 590nanometer emission.

A cytotoxic EC₅₀ is derived by plotting compound concentration on thex-axis and average percent inhibition of cell growth for each titrationpoint on the y-axis. Growth of cells in control wells that have beentreated with vehicle alone is defined as 100% growth for the assay, andthe growth of cells treated with compounds is compared to this value.Proprietary in-house software is used to calculate percent cytotoxicityvalues and inflection points using logistic 4-parameter curve fitting.Percent cytotoxicity is defined as:%cytotoxicity:(Fluorescence_(control))−(Fluorescence_(sample))×100×(Fluorescence_(control))⁻¹The inflection point is reported as the cytotoxic EC₅₀.III. Evaluation of Mitotic Arrest and Apoptosis by FACS

FACS analysis is used to evaluate the ability of a compound to arrestcells in mitosis and to induce apoptosis by measuring DNA content in atreated population of cells. Cells are seeded at a density of 1.4×10⁶cells per 6 cm² tissue culture dish and allowed to adhere overnight.Cells are then treated with vehicle (0.1% DMSO) or a titration series ofcompound for 8-16 hours. Following treatment, cells are harvested bytrypsinization at the indicated times and pelleted by centrifugation.Cell pellets are rinsed in PBS and fixed in 70% ethanol and stored at 4°C. overnight or longer.

For FACS analysis, at least 500,000 fixed cells are pelleted and the 70%ethanol is removed by aspiration. Cells are then incubated for 30 min at4° C. with RNase A (50 Kunitz units/ml) and propidium iodide (50 μg/ml),and analyzed using a Becton Dickinson FACSCaliber. Data (from 10,000cells) is analyzed using the Modfit cell cycle analysis modelingsoftware (Verity Inc.).

An EC₅₀ for mitotic arrest is derived by plotting compound concentrationon the x-axis and percentage of cells in the G2/M phase of the cellcycle for each titration point (as measured by propidium iodidefluorescence) on the y-axis. Data analysis is performed using theSigmaPlot program to calculate an inflection point using logistic4-parameter curve fitting. The inflection point is reported as the EC₅₀for mitotic arrest. A similar method is used to determine the compoundEC₅₀ for apoptosis. Here, the percentage of apoptotic cells at eachtitration point (as determined by propidium iodide fluorescence) isplotted on the y-axis, and a similar analysis is carried out asdescribed above.

IV. Immunofluorescence Microscopy to Detect Monopolar Spindles

Methods for immunofluorescence staining of DNA, tubulin, and pericentrinare essentially as described in Kapoor et al. (2000) J. Cell Biol. 150:975-988. For cell culture studies, cells are plated on tissue culturetreated glass chamber slides and allowed to adhere overnight. Cells arethen incubated with the compound of interest for 4 to 16 hours. Afterincubation is complete, media and drug are aspirated and the chamber andgasket are removed from the glass slide. Cells are then permeabilized,fixed, washed, and blocked for nonspecific antibody binding according tothe referenced protocol. Paraffin-embedded tumor sections aredeparaffinized with xylene and rehydrated through an ethanol seriesprior to blocking. Slides are incubated in primary antibodies (mousemonoclonal anti-α-tubulin antibody, clone DM1A from Sigma diluted 1:500;rabbit polyclonal anti-pericentrin antibody from Covance, diluted1:2000) overnight at 4° C. After washing, slides are incubated withconjugated secondary antibodies (FITC-conjugated donkey anti-mouse IgGfor tubulin; Texas red-conjugated donkey anti-rabbit IgG forpericentrin) diluted to 15 μg/ml for one hour at room temperature.Slides are then washed and counterstained with Hoechst 33342 tovisualize DNA. Immunostained samples are imaged with a 100× oilimmersion objective on a Nikon epifluorescence microscope usingMetamorph deconvolution and imaging software.

1. The compound of the Formula III:

wherein: R¹ is selected from: H, (C═O)_(a)(C₁-C₆)alkyl,(C₁-C₆)alkyl-phenyl, (C₃-C₈)cycloalkyl and (C═O)N(R^(d))₂; R³ is H or(C₁-C₆)alkyl; R⁵ is halogen; R^(d) is independently selected from H or(C₁-C₆)alkyl; p is 0; n is 2; or a pharmaceutically acceptable salt orstereoisomer thereof.
 2. A pharmaceutical composition comprising apharmaceutical carrier, and dispersed therein, a therapeuticallyeffective amount of a compound of claim
 1. 3. The compound of claim 1which is selected from:2-benzyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole;2-cyclohexyl-5-(2,5-difluorophenyl)-3-phenyl-2,3-dihydroisoxazole;5-(2,5-difluorophenyl)-2-isopropyl-3-phenyl-2,3-dihydroisoxazole;5-(2,5-difluorophenyl)-2-methyl-3-phenyl-2,3-dihydroisoxazole;5-(5-chloro-2-fluorophenyl)-N,3-dimethyl-N-(1-methylpiperidin-4-yl)-3-phenylisoxazole-2(3H)-carboxamide;2-acetyl-5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole;5-(5-chloro-2-fluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole;5-(5-chloro-2-fluorophenyl)-2-(2,2-dimethylpropanoyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole;5-(5-chloro-2-fluorophenyl)-N-[3-(dimethylamino)propyl]-N,3-dimethyl-3-phenylisoxazole-2(3H)-carboxamide;and2-acetyl-5-(2,5-difluorophenyl)-3-methyl-3-phenyl-2,3-dihydroisoxazole;or a pharmaceutically acceptable salt or stereoisomer thereof.